酶解制备苦荞蛋白抗氧化肽及其分离纯化研究

以苦荞麦粉为原料,提取苦荞蛋白,分别采用碱性蛋白酶、胃蛋白酶、胰蛋白酶对蛋白进行酶解,采用DPPH法比较不同酶解产物的抗氧化活性,从而筛选水解制备苦荞蛋白抗氧化肽的最适酶。以水解度为指标,利用单因素试验和响应面法优化酶解工艺条件。结果表明,不同蛋白酶酶解产物的抗氧化活性大小为:胃蛋白酶胰蛋白酶碱性蛋白酶,其中胃蛋白酶酶解产物的DPPH自由基清除率最高,为68.47%。胃蛋白酶最佳水解工艺条件为:时间2.5 h、温度38℃、pH 2.0,在此条件下苦荞蛋白水解度为32.68%。采用超滤对苦荞蛋白水解物进行分离纯化,结果表明,分子量3 kDa的水解物具有显著的抗氧化活性;经凝胶过滤色谱进一步分离得到3个峰,小分子量峰组分显示出最强的抗氧化活性。     

复合酶解牦牛乳酪蛋白工艺优化及抗氧化性的研究

为了优化牦牛乳酪蛋白酶解工艺,研究其产物抗氧化活性,选用中性蛋白酶、碱性蛋白酶、胃蛋白酶、胰蛋白酶和木瓜蛋白酶,在其最适条件下酶解牦牛乳酪蛋白,以水解度(DH)、DPPH·自由基清除率和超氧阴离子清除率为评价指标,筛选出2种效果最优的单酶进行复配,采用单因素试验及L9(34)正交试验确定最优酶解工艺。结果表明:中性蛋白酶和胰蛋白酶比例为1:2效果最佳,最佳酶解工艺条件为底物浓度5%(w/v),温度42.5℃,p H值7.5,复合酶添加量3%,作用时间150 min,酶解产物抗氧化活性最高,DPPH·自由基清除率达到了64.26%±0.18%,超氧阴离子清除率达到40.34%±0.92%。     

不同酶解法水解黑龙江小麦麦胚蛋白的抗氧化功能比较研究

通过采用四种不同蛋白酶对麦胚蛋白分别进行单酶水解、双酶同步水解和分步水解,以水解产物的水解度和抗氧化性为指标,比较研究麦胚蛋白的酶解方法与水解物的抗氧化功能的关系。结果表明：单酶水解时碱性蛋白酶的水解物抗氧化效果最好,DPPH 自由基清除率达到39.74%;双酶分步水解的效果优于双酶同步水解,其中先加碱性蛋白酶后加木瓜蛋白酶效果最好,DPPH 自由基清除率达到45.36%。因此选择先加碱性蛋白酶后加木瓜蛋白酶作为水解麦胚蛋白最佳工艺。     

木瓜蛋白酶和碱性蛋白酶分步水解玉米蛋白酶解产物抗氧化活性研究

采用木瓜蛋白酶和碱性蛋白酶水解玉米蛋白粉,在两种酶最佳作用条件下,对两种酶作用时间进行了研究,结果表明,采用木瓜蛋白酶和碱性蛋白酶双酶分步水解其酶解产物的还原力和DPPH·清除率显著提高。木瓜蛋白酶水解1 h后再用碱性蛋白酶水解2 h得到玉米蛋白酶解产物的抗氧化效果最好,且其酶解产物的还原力为0.725,DPPH·清除率为69.6%。     

不同蛋白酶对小麦蛋白酶解物抗氧化活性的影响

小麦蛋白是小麦淀粉加工的副产物,酶解是提高小麦蛋白溶解性和功能性的有效方式,而酶解用酶种类可能对酶解产物的功能性如抗氧化活性有一定影响。采用碱性蛋白酶、中性蛋白酶、胃蛋白酶、风味蛋白酶、胰蛋白酶、木瓜蛋白酶6种常用的蛋白酶分别对小麦蛋白进行酶解,并对酶解4 h后酶解物的多肽得率、分子质量分布、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率、超氧阴离子自由基(O_2~-·)清除率、羟自由基(·OH)清除率等反映水解程度和抗氧化能力的主要指标进行评价。结果表明,风味蛋白酶酶解物中多肽得率最高,达91.44%,且分子质量小于3 000 D的多肽含量达76.9%;酶解物质量浓度为3 mg/m L时,木瓜蛋白酶酶解物对DPPH自由基清除作用最好,清除率为65.12%(P0.01),其次是风味蛋白酶(58.43%)和碱性蛋白酶(55.29%);碱性蛋白酶酶解物对O_2~-·清除率效果最好,清除率为58.68%(P0.01),其次是风味蛋白酶(49.25%);碱性蛋白酶和木瓜蛋白酶酶解物对·OH清除效果最佳,清除率分别为59.23%和58.16%。结果说明,蛋白酶种类对小麦蛋白酶解物抗氧化活性影响显著,风味蛋白酶对提高蛋白水解程度和生成小分子质量多肽的作用明显,而碱性蛋白酶、木瓜蛋白酶和风味蛋白酶对提高酶解产物抗氧化活性效果较好。     

二步酶解法制备鱼鳞明胶抗氧化肽及其抗氧化活性研究

通过碱性蛋白酶进行第一步酶解,胰蛋白酶、风味蛋白酶、木瓜蛋白酶和胃蛋白酶分别进行第二步酶解制备鱼鳞明胶抗氧化肽。结果表明,二步酶解法能有效提高酶解物的水解度,降低水解物的分子量,碱性蛋白酶—胰蛋白酶水解物具有显著的自由基清除能力和Fe~(2+)螯合能力。通过响应面优化的胰蛋白酶最佳二步酶解工艺为:底物浓度100 mg/mL、pH 7.8、温度53℃、时间50min,该条件下制备的水解物的Fe~(2+)螯合率为65.72%,清除DPPH·、·OH和O_2~-·的IC_(50)值分别是7.39,0.68,1.84mg/mL。     

蛋白酶种类及水解时间对猪血浆蛋白水解物抗氧化性和乳化性的影响

主要探讨蛋白酶种类及水解时间对猪血浆蛋白水解物抗氧化活性以及乳化能力的影响。采用3种蛋白酶(碱性蛋白酶、木瓜蛋白酶和中性蛋白酶)对猪血浆蛋白分别水解20、40、60、80、120 min。测定猪血浆蛋白水解物的抗氧化活性、乳化活力、乳化稳定性以及分子质量的变化趋势。结果表明:相对于木瓜蛋白酶和中性蛋白酶来说,碱性蛋白酶能够显著提高猪血浆蛋白水解物的还原能力、ABTS~+·和1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除能力(P0.05),而且碱性蛋白酶水解60 min时所获得的猪血浆蛋白水解物具有最高的乳化活力和乳化稳定性(P0.05)。另外,随着水解时间的延长,猪血浆蛋白水解物中小于5 k D的成分显著增加(P0.05),且碱性蛋白酶对猪血浆蛋白的酶解效果最佳(P0.05)。上述结果表明,碱性蛋白酶适度水解猪血浆蛋白以后获得的水解物同时具有良好的抗氧化活性和乳化能力。     

酶法水解滑菇蛋白制备抗氧化肽的工艺优化

本文研究了酶法水解滑菇蛋白制备抗氧化肽的工艺条件,通过水解度与羟自由基(·OH)清除率来综合评价水解产物的抗氧化能力。以·OH清除率为指标,从碱性蛋白酶、中性蛋白酶、风味蛋白酶、木瓜蛋白酶和胰蛋白酶5种蛋白酶中筛选最适酶。在单因素实验研究酶浓度、底物浓度与水解时间对酶解产物·OH清除率和水解度影响的基础上,通过L_9(4~3)正交实验对水解工艺进行优化。结果得出,碱性蛋白酶活力高于其他4种蛋白酶,能使蛋白质充分水解,正交实验各因素对水解产物·OH清除率的影响程度依次为底物浓度酶浓度水解时间。其中,碱性蛋白酶的最佳水解工艺条件为:底物浓度3.0%、酶浓度3500 U/g、p H9.0、温度55℃、水解时间3.5 h。在此条件下,水解产物的·OH清除率可达82.7%,水解度为23.1%。碱性蛋白酶水解滑菇蛋白制备抗氧化肽的工艺可行,为滑菇蛋白高附加值产品的开发提供技术参考。     

鸡骨酶解物的抗氧化活性研究

分别选用复合风味蛋白酶、Protamex复合蛋白酶、Alcalase蛋白酶、胰酶、胰蛋白酶、AS.1398中性蛋白酶和木瓜蛋白酶水解鸡骨泥,研究鸡骨酶解物(蛋白质含量3 mg/mL)对DPPH、羟自由基(OH·)的清除能力及其还原性.试验结果表明:Protamex复合蛋白酶酶解鸡骨产物的DPPH清除率最强,即81.44%;复合风味蛋白酶酶解物OH·清除率达30.32%,还原性测定的吸光值为0.3.总之,鸡骨酶解物的DPPH清除能力强于羟自由基清除能力和还原力.在研究不同浓度和水解度对鸡骨酶解物清除DPPH活性的影响时发现,随着酶解物蛋白质质量浓度的增大,酶解物的DPPH清除率升高.复合风味蛋白酶、Protamex复合蛋白酶的鸡骨酶解物蛋白质质量浓度分别为4、6mg/mL时,其DPPH清除率分别达91.49%、97.3%.但DPPH清除率与水解度不呈正相关性,只有在特定水解度下,使抗氧化肽含量最高时,水解物才表现出最强的抗氧化能力.例如复合风味蛋白酶、AS.1398中性蛋白酶、Protamex复合蛋白酶、胰酶的最佳水解度分别为12.59%、23.78%、14.45%、20.04%.     

不同蛋白酶对碧根果蛋白肽制备的影响及抗氧化活性研究

以碧根果为原料，用碱提酸沉法从碧根果中制备碧根果蛋白；采用木瓜蛋白酶、中性蛋白酶、胃蛋白酶、胰蛋白酶、碱性蛋白酶水解碧根果蛋白以制备蛋白肽。考察不同蛋白酶对碧根果蛋白水解程度、理化特性和抗氧化活性的影响，旨在筛选出适合的蛋白酶类型制备碧根果抗氧化肽。结果表明，碱性蛋白酶水解碧根果蛋白6 h时水解度最高[（32.54±1.21）%]，此外，不同蛋白酶处理使碧根果蛋白的疏水性氨基酸暴露，使红外光谱发生红移，内源荧光强度增强。抗氧化实验结果表明碱性蛋白酶蛋白肽具有较好的DPPH自由基清除能力，木瓜蛋白酶蛋白肽具有较好的·OH和ABTS+自由基清除能力。本研究为碧根果蛋白的后期的深加工利用提供一定理论依据。     

银鲳酶解物抗氧化活性研究

选用胃蛋白酶、胰蛋白酶、碱性蛋白酶和中性蛋白酶对银鲳蛋白进行酶解以制备蛋白酶解物,以羟基自由基清除活性为指标确定银鲳最佳水解酶。结果显示,碱性蛋白酶的水解物抗氧化活性最强。实验对碱性蛋白酶水解银鲳的酶解条件(时间、温度、pH、酶添加量和固液比)进行正交实验设计,并对最佳水解条件下所获得的酶解物进行抗氧化活性测试。结果表明,银鲳蛋白碱性蛋白酶水解物对DPPH自由基和羟基自由基具有清除作用,其自由基清除效果呈现剂量依赖性,而且银鲳蛋白水解物还具有明显还原能力。所有这些体外抗氧化数据说明,银鲳蛋白水解物有明显的抗氧化效力。     

大豆蛋白的酶水解及类蛋白反应对其抗氧化活性的影响

采用Alcalase 2.4L FG 蛋白酶水解大豆蛋白,筛选并制备出ABTS+·清除率最高的水解物,其水解度为14.0%,对ABTS+·清除率为43.6%。以此水解物为底物,以修饰产物的游离氨基减少量为指标,应用响应面分析得到类蛋白反应的优化条件为：酶添加量1037U/g pro、底物质量浓度30g/100mL、温度20℃。在此条件下反应6h,水解物的修饰反应程度和抗氧化活性均为最大。制备反应程度不等的3 个修饰产物,进一步抗氧化活性分析表明：大豆蛋白水解物及其修饰产物的抗氧化活性好于大豆蛋白;修饰产物与水解物的DPPH 自由基清除能力、还原力、超氧阴离子自由基(O2 － ·)清除率差别不显著,但是对羟自由基(·OH)清除率差别显著。     

Antioxidant properties and protein compositions of porcine haemoglobin hydrolysates

Porcine haemoglobin hydrolysates were prepared through hydrolysis by Alcalase followed by Flavourzyme, and their protein compositions were analyzed using Sephadex G-50 gel filtration chromatography. The antioxidant activities, including reducing power, ferrous ion chelating ability, and DPPH radical scavenging activity, of the hydrolysates were evaluated. The results showed that the hydrolysates of haemoglobin exhibited low reducing powers, but high ferrous ion chelating abilities and DPPH radical scavenging activities. The hydrolysate, obtained through hydrolysis by 2% Alcalase for 4 h and followed by 1% Flavourzyme for 6 h, had the highest ferrous ion chelating ability of 63.54% at a concentration of 5.0 mg/mL. The hydrolysate, obtained through hydrolysis by 2% Alcalase for 4 hrs, had the highest DPPH radical scavenging activity of 41.94% at a concentration of 5.0 mg/mL. According to the results of protein composition analysis, we divided the hydrolysates into three groups, including high molecular weight (MW) group (Group I), medium MW group (Group II), and low MW group (Group III). The reducing power and ferrous ion chelating ability of the hydrolysates were significantly and positively correlated to the relative amount of Group I, and negatively correlated to the relative amount of Group III. This study revealed that the antioxidant activities of porcine haemoglobin hydrolysates were dependent on their protein compositions. The high MW protein fraction (Group I) was responsible for the high reducing power and ferrous ion chelating ability of the hydrolysate.     

Effect of defatting and enzyme type on antioxidative activity of shrimp processing byproducts hydrolysate

Shrimp processing byproducts (SPB) was digested by 6 proteases (trypsin, pepsin, neutrase, Protamex, Flavourzyme, and Alcalase) to produce antioxidative peptides. Both degree of hydrolysis (DH) and DPPH radical scavenging activity (DSA) of the Alcalase hydrolysate were the highest of all. The effect of defatting on DH and DSA of the Alcalase hydrolysate was significant. The DH decreased while the DSA increased after defatting of the byproducts. The antioxidative activity of Alcalase hydrolysate was also investigated using several in vitro assays, including DPPH, ABTS radical scavenging assays (ASA), reducing power assay, and chelating activity. The antioxidative activity of the hydrolysate was obviously concentration dependent. The SPB Alcalase hydrolysate exhibited notable DSA and ASA with the IC50 values of 500 and 7.4 μg/mL, respectively. And the hydrolysate showed 38.9% chelating activity at 120 μg/mL level. The SPB Alcalase hydrolysate was a potential source of natural antioxidants.     

Amino acid,structure and antioxidant properties of Haematococcus pluvialis protein hydrolysates produced by different proteases

The impact of different protease hydrolysis on the amino acid, structure and antioxidant properties of H. pluvialis protein (HP) was investigated. Results showed that the hydrolysate obtained by Alcalase exhibited the highest degree of hydrolysis (20.59%) and peptide yield (92.64%). The essential amino acid, hydrophobic, sulphur and aromatic amino acid contents of enzyme hydrolysates were significantly higher than HP (P < 0.05). FTIR spectra showed that the β-sheet proportion of HP hydrolysates were higher compared with HP, the proportion of random coil structure was lower. The α-helix content of the hydrolysate obtained by Alcalase was the highest, while the turn proportion was the lowest. The Trypsin derived hydrolysate presented the best DPPH and ABTS scavenging ability, and ferric reducing antioxidant power than other HPHs. These results suggested that HP hydrolysates have a great potential as natural functional ingredients in food manufacture.     

花椒籽仁抗氧化肽水解用酶的筛选研究

采用Protamex、Alcalase2.4L、Flavourzyme500MG、Neutrase0.5L、木瓜蛋白酶5种酶制剂水解花椒籽仁蛋白制备抗氧化肽,以水解度(DH)、多肽含量和水解产物的总抗氧化能力(TAC)、DPPH自由基清除能力、在亚油酸体系中的抗氧化性为指标对水解过程进行了分析,结果表明,Alcalase2.4L蛋白酶是制备花椒籽仁抗氧化肽的最适水解酶,其水解物的水解度(DH)为20.42%,多肽含量为21.83mg/mL,总抗氧化能力(TAC)和DPPH自由基清除能力分别为0.44mmol/L、65.47%,此外在亚油酸体系中具有一定的抗氧化性。     

Antioxidant activity of several marine skin gelatins

Gelatins obtained from the skins of tuna and halibut, and from the tunics of jumbo flying squid were hydrolysed by Alcalase to produce antioxidant peptides. Hydrolysis yielded an increase in the antioxidant capacity of gelatins of around two-fold when measured by the Fe reducing capacity (FRAP) method and even more when measured by the ABTS radical scavenging method. When both squid and tuna gelatins were hydrolysed with different enzymes (collagenase, trypsin, pepsin), Alcalase and pepsin gave the hydrolysates with the highest and lowest ABTS radical scavenging ability, respectively. FRAP assay showed that the squid hydrolysates prepared using Alcalase were the most effective in reducing ferric ions, whereas trypsin gave rise to the tuna hydrolysates with the highest iron reducing ability. When the amino acid composition of the gelatins was related to the antioxidant properties, ABTS radical scavenging was observed to be negatively correlated to the total content of hydrophobic amino acids and imino acids in all the samples, while Fe reducing power (FRAP) was strongly correlated with Hyl and degree of hydroxylation.     

2种蛋白酶酶解曲拉干酪素条件优化及抗氧化性比较

以曲拉干酪素为原料、水解度为指标,在酶解时间、酶解温度、pH值、曲拉干酪素质量浓度、酶添加量单因素试验基础上,采用响应面试验对碱性蛋白酶和胰蛋白酶酶解工艺条件进行优化,并对2 种酶解液的1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、超氧阴离子自由基、羟自由基清除率,Fe2＋、Cu2＋螯合能力和还原力等抗氧化性指标进行比较。结果表明,碱性蛋白酶和胰蛋白酶分别在酶解时间3.8、2.5 h,酶解温度49.8、47.8 ℃,曲拉干酪素质量浓度60、35 g/L,pH 8.5、7.5,酶添加量140、2 900 U/g时水解度最大,为24.25%和13.57%。碱性蛋白酶解液超氧阴离子自由基清除率、Fe2＋螯合能力显著低于胰蛋白酶解液（P＜0.01）;羟自由基清除能力高于胰蛋白酶解液（P＞0.05）;2 种蛋白酶酶解液在酶解液质量浓度1～5 mg/mL时,Cu2＋螯合能力、DPPH自由基清除率和还原力随质量浓度均呈上升趋势,Cu2＋螯合能力低于Fe2＋螯合能力（P＞0.05）,DPPH自由基清除率和还原力二者差异显著（P＜0.01）。2 种蛋白酶对酶解物抗氧化性指标影响不同,碱性蛋白酶酶解物抗氧化性相对较优。     

Functionalities and antioxidant properties of protein hydrolysates from the muscle of ornate threadfin bream treated with pepsin from skipjack tuna

Functional properties and antioxidant activities of protein hydrolysates prepared from ornate threadfin bream (Nemipterus hexodon) muscle, using skipjack tuna pepsin, with different degrees of hydrolysis (DH: 10%, 20% and 30%), were evaluated. Emulsifying and foaming properties of hydrolysates were governed by their DH and concentrations used. Hydrolysates with 20% DH had the highest scavenging activities for ABTS and DPPH radicals. However, chelating activity of hydrolysates for ferrous ion increased as DH increased. Size exclusion chromatography of the hydrolysate with 20% DH using Sephadex G-25 revealed that antioxidative peptides with molecular weight of approximately 1.3 kDa exhibited the highest ABTS radical-scavenging activity. In vitro simulated gastrointestinal digestion indicated that ABTS radical-scavenging activity of the antioxidative peptides was not affected by pepsin hydrolysis, whilst further digestion by pancreatin enhanced the activity. Therefore, protein hydrolysate from the muscle of ornate threadfin bream produced by skipjack tuna pepsin can be used as a promising source of functional peptides with antioxidant properties.